Lignin is one of the major products of the phenylpropanoid pathway, and is one of the most abundant organic molecules in the biosphere (Crawford, (1981) Lignin Biodegradation and Transformation, New York: John Wiley and Sons). During lignin biosynthesis, caffeic acid is believed to be O-methylated by caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) to form ferulic acid, a direct precursor of lignin. COMT is also believed to play a role in the final hydroxylation reaction of the general phenylpropanoid pathway, in which it O-methylates 5-hydroxyferulate. This dual specificity of COMT has been confirmed by the cloning of the COMT gene, and expression of the protein in E. coli (Bugos et al., Plant Mol. Biol. 17, 1203, (1991); Gowri et al., (1991) Plant Physiol., 97, 7, (1991)). The expression pattern of the COMT gene and the evidence from mutations of the COMT gene are consistent with this role in lignification (Marita et al., J. Chem. Soc., Perkin Trans. 1, 2939-2945 (2001). Accordingly, COMT is believed to play an important role in lignin biosynthesis, and control of COMT expression may provide a means for regulating lignin biosynthesis and other processes that occur concurrently in cell wall development, such as formation of hemicellulose and lignocellulosic polymers.
Lignin biosynthesis can be regulated at the level of transcription. During transcription, a single-stranded RNA complementary to the DNA sequence to be transcribed is formed by the action of RNA polymerases. Initiation of transcription in eucaryotic cells is regulated by complex interactions between cis-acting DNA motifs, located upstream of the gene to be transcribed, and trans-acting protein factors. Among the cis-acting regulatory elements are sequences of DNA, termed promoters, which are located close to the transcription initiation site and to which RNA polymerase is first bound, either directly or indirectly. Promoters usually consist of proximal (e.g. TATA box) and more distant elements (e.g. CCAAT box). Enhancers are cis-acting DNA motifs which may be situated 5-prime and/or 3-prime from the initiation site.
Both promoters and enhancers are generally composed of several discrete, often redundant, elements each of which may be recognized by one or more transacting regulatory proteins, known as transcription factors. Regulation of the complex patterns of gene expression observed both spatially and temporally, in all developing organisms, is thought to arise from the interaction of enhancer- and promoter-bound, general and tissue-preferred transcription factors with DNA (Izawa T, Foster R and Chua N H, 1993, J. Mol. Biol. 230:1131-1144; Menkens A E, Schindler U and Cashmore A R, 1995, Trends in Biochem Sci 13:506-510).
Genetic regulation of biochemical pathways may be conducted in narrowly restricted tissue types to avoid global, detrimental effects to the modified plants. For example, when the content or composition of lignin is affected by expression of a particular gene product, it may be desirable to limit the expression of the gene product to certain segments of the plant or to certain developmental stages, to avoid decreasing the plant's disease resistance. A heterologous gene may be expressed in a selected tissue by operably linking it to a tissue-preferred promoter. Suitable tissue-preferred promoters include the bean grp 1.8 promoter, which is specifically active in protoxylem tracheary elements of vascular tissue. Keller et al., EMBO J. 8: 1309 (1989). These promoters also include the Eucalyptus CAD promoter, which is preferentially expressed in lignifying zones. Feuillet et al., Plant Mol. Biol. 27: 651 (1995). Such tissue-preferred promoters have been used to regulate gene expression of antisense molecules in specific tissues. Van der Meer et al., Plant Cell 4: 253 (1992), Salehuzzaman et al., Plant Mol. Biol. 23: 947 (1993), and Matsuda et al., Plant Cell Physiol. 37: 215 (1996).
Because tissue-preferred promoters may be less active in a heterologous environment, they do not always express genes to the same levels achieved with constitutive promoters. Yahiaoui et al., Phytochemistry 49: 295-306 (1998). Further, the developmental window during which these promoters are active, or the spatial distribution of their activity, may limit their usefulness. Thus, there is a continuing need in the art for additional tissue-preferred promoters, especially vascular-preferred promoter sequences, that have desirable spatial and temporal patterns of expression. Grima-Pettenati et al., Plant Science 145: 51-65 (1999).
WO 01/98485 and U.S. Ser. No. 10/137,036 disclose a full-length Eucalyptus grandis COMT promoter and 661 base pair fragment. However, neither application suggests that other deletion fragments of the Eucalyptus grandis COMT promoter are active as promoters in and of themselves, or that such deletion fragments might confer tissue-preferred, tissue-specific, or vascular-preferred expression.